Method of using catalpic acid to treat type 2 diabetes and associated disorders

ABSTRACT

A method of treating and preventing type 2 diabetes and obesity an animal, including mammals and humans, in which a therapeutically effective amount of catalpic acid to the animal is administered orally or parentally.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application Ser. No. 60/644,284 filed 14 Jan. 2005, theentirety of which is incorporated herein.

FIELD OF THE INVENTION

The present invention is generally directed to a method of usingcatalpic acid to treat and prevent metabolic disorders in animals,including mammals and humans. More specifically, a method is providedusing catalpic acid to normalize impaired glucose tolerance, to preventhyperglycemia and hyperinsulinemia, improve glucose tolerance and type 2diabetes, to attenuate the detrimental effects of high fat diets and tominimize abdominal fat accumulation.

CITED REFERENCES

A full bibliographic citation of the references cited in thisapplication can be found in the section preceding the claims.

DESCRIPTION OF THE PRIOR ART

A need exists for novel methods of treating or preventing metabolicdisorders such as the Metabolic Syndrome or Syndrome X, obesity and type2 diabetes. In western societies, the high prevalence of obesity resultsin several metabolic disorders such as type 2 diabetes, cardiovasculardisease, hypertension, and hyperlipemia, which are characterized asMetabolic Syndrome or Syndrome X (Wang et al. 2003). The MetabolicSyndrome has an estimated age-adjusted US prevalence of 23.7% (Ginsberg2003). According to estimates from the Centers for Disease Control(“CDC”) and Prevention published in the National Diabetes Fact Sheet,the number of Americans afflicted by diabetes increased from 5.8 millionin 1980 to 13.3 million in 2002, representing 6.3% of the populationwith 1 million newly diagnosed cases and $132 billion in medicalexpenses per year (CDC 2004). Treatments against the Metabolic Syndrometarget the disease signs and not the cause. For instance, aspirin isrecommended for its suppressive effect on platelet aggregation, andbeta-blockers are utilized for treating diabetic hypertension and toreduce the mortality associated with cardiovascular disease. Noveltreatments for insulin resistance include agonists of peroxisomeproliferator-activated receptor gamma such as thiazolidinediones (TZDs),whereas statins and fibrates are utilized to favorably modify the bloodlipid profiles and treat the dylipidemia found in Metabolic Syndromepatients (Ginsberg 2003). However, questions have been raised regardingthe safety of TZD-based treatments due to adverse cardiovascular (fluidretention and congestive heart failure) and liver (fatty liver) sideeffects. Therefore, there remains a need to identify novel and safermethods of preventing or treating the Metabolic Syndrome, type 2diabetes, obesity and their complications, including nutritional methodsthat act upon molecular networks located in the interface betweenimmunity, inflammation and metabolism.

Catalpic acid is a non-toxic, natural, orally active compound. Catalpicacid is naturally found in seeds of some ornamental trees, i.e., Catalpaovata (Chinese Catalpa), Catalpa speciosa (Northern Catalpa), Catalpabungei, or Catalpa bigninioides, representing 40 to 70 percent of theoil. The presence of catalpic acid in the seeds of catalpa trees iswell-known in the field. Also, the capacity of triglyceride esters ofcatalpic acid to serve as drying oils in the fabrication of primers oradhesion or sealing compositions is well-known in the field. Forinstance, U.S. Pat. No. 6,451,439 to Okamoto teaches a method ofeffecting adhesion for sealing compositions. However, this method doesnot teach the use of catalpa oil or catalpic acid to treat or preventmetabolic disorders, such as type 2 diabetes, obesity or the MetabolicSyndrome.

U.S. Pat. No. 6,593,514 to Cahoon teaches a method for the production ofcalendic acid, a fatty acid containing delta-8, 10, 12 conjugated doublebonds and related fatty acids having a modification at the delta-9position. While the patent lists catalpic acid in a long list ofconjugated trienes, this method does not teach the use of catalpa oil orcatalpic acid to treat and prevent metabolic disorders, such as type 2diabetes, obesity or the Metabolic Syndrome.

U.S. Patent Application 20030126640 to Cahoon teaches a method forpreparing and using nucleic acid fragments encoding plant fatty acidmodifying enzyme associated with conjugated double bond formation orfunctionally equivalent subfragments thereof. This method could beutilized to create transgenic plants producing altered lipid profiles.These altered lipid profiles could include catalpic acid and otherconjugated trienes. However, this method does not teach the use of theseconjugated trienes in general or catalpic acid in particular in theprevention and treatment of metabolic disorders, such as type 2diabetes, obesity or the Metabolic Syndrome.

U.S. Patent Application 20020045232 to Qiu teaches a method forlarge-scale production of conjugated fatty acids, especially conjugatedlinoleic and linolenic acids in plants. The invention relates to genesidentified from Calendula officinalis coding for a conjugase and itsrelated enzyme, a DELTA.12 desaturase, and utilization of them for largescale production of conjugated linoleic and linolenic acids in plants.The constructs containing these genes can be transferred to plants withdifferent substrate profiles, which allows for the production ofconjugated linoleic acids (18:2, DELTA.8, DELTA.10) and linolenic acids(DELTA.8, DELTA.10, DELTA.12) in plant seeds on a commercial scale.However, this method does not teach the use of conjugated linoleic orlinolenic acids in general or catalpic acid in particular in theprevention and treatment of metabolic disorders, such as type 2diabetes, obesity or the Metabolic Syndrome.

It is therefore an object of the present invention to provide a methodof treating and preventing type 2 diabetes and obesity of an animal,including mammals and humans, using catalpic acid.

It is still another object of the present invention to provide a methodof treating and preventing the impaired glucose tolerance, thehyperglycemia, the hyperinsulinemia and the excessive abdominal fataccumulation found in type 2 diabetes and obesity by using catalpicacid.

It is still another object of the present invention to provide a methodto ameliorate the detrimental metabolic changes associated with theconsumption of a meal containing a high concentration of fat by usingcatalpic acid.

It is another object of the present invention to provide a method oftreating and preventing the complications derived from insulinresistance, type 2 diabetes and obesity such as the ischemic diabeticfoot syndrome, hypertension, nephropathy, neuropathy, retinopathy,polycystic ovaries, and cardiovascular disease, including coronary heartdisease, endothelial dysfunction and myocardial infarction by usingcatalpic acid.

SUMMARY OF THE INVENTION

In response to the above-described needs, the present invention providesa method of treating and preventing insulin resistance, type 2 diabetesand obesity in an animal, including mammals and humans, in need thereof.The method comprises administering a therapeutically effective amount ofa compound selected from the group consisting of catalpic acid, estersthereof, pharmaceutically-suitable salts thereof, metabolites thereof,and combinations thereof.

In addition, the present invention provides a composition for treatingtype 2 diabetes and abdominal obesity signs comprising an effectiveamount of a compound selected from the group consisting of catalpicacid, non-toxic salts thereof, active esters thereof, structural lipidscontaining catalpic acid, methyl and ethyl esters thereof, activemetabolites thereof and other active chemical derivatives thereof andmixtures thereof, in combination with a pharmaceutically-acceptablecarrier.

The compound may be administered to the animal in a single dose or inmultiple doses. This method utilizes the natural qualities of catalpicacid to treat and prevent type 2 diabetes and obesity in an animal,including mammals and humans. Specifically, an amount effective tonormalize impaired glucose tolerance, prevent hyperglycemia, preventhyperinsulinemia, and minimize abdominal fat accumulation isadministered. While any of the catalpic acid forms may be used, in apreferred embodiment, the free acid form of catalpic acid is used.

In a preferred embodiment of the present invention, the catalpic acidcompound is administered orally to the animal. The catalpic acidcompound may also be administered parenterally, via injection orrectally. The catalpic acid compound may be administered alone or incombination with a pharmaceutically suitable carrier or excipient.

In another embodiment of the present invention, a therapeuticallyeffective amount of the catalpic acid compound is administered to ananimal in combination with a nutritional food supplement. Suchsupplements include but are not limited to infant formulas, childrenproducts, geriatric formulas, milk, cheese, kefir, cereal bars, weightmanagement formulas, energy bars, other human foods, functional foods,and animal feed. Catalpic acid may also be administered in combinationwith other active ingredients such as vitamins or other fatty acids.

The effective amount of the catalpic acid compound depends on the needsof the animal. The formulations of catalpic acid disclosed in thepresent invention may be conveniently presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy ornutrition. Possible formulations include but are not limited tocapsules, cachets, tablets, boluses or lozenges, each containing apredetermined amount of catalpic acid.

There are many advantages to the method of the present invention. Forinstance, there is no upper limit on the amount of catalpic acid thatmay be administered to an animal in need thereof. Further, the method ofthe present invention may be administered to animals, including mammalsand humans, of all ages and health. For instance, vulnerable populationssuch as the elderly, obese, diabetic, sick or very young can benefitfrom the present invention, as can healthy individuals with no historyof chronic disease. In addition, the method of the present invention maybe administered in a variety of ways, thereby providing a versatile andefficient means of preventing metabolic disorders in a mammal.

The objects and advantages of the invention will appear more fully fromthe following detailed description of the preferred embodiment of theinvention made in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph illustrating the effect of catalpic acid on bloodglucose concentrations during a glucose tolerance test in mice fedregular diets from Experiment 1.

FIG. 1B is a graph illustrating the effect of catalpic acid on bloodglucose concentrations during a glucose tolerance test in mice fed highfat diets from Experiment 1.

FIG. 2A is a graph illustrating the effect of dietary catalpicacid-supplementation on fasting plasma glucose concentrations on days 0,7, 14, 21 and 28 of Experiment 2.

FIG. 2B is a graph illustrating the effect of dietary catalpicacid-supplementation on plasma glucose concentrations during a glucosetolerance test from Experiment 2.

FIG. 3 is a graph illustrating the effect of dietary catalpicacid-supplementation on fasting plasma insulin concentrations on days 0,7, 14 and 28 of Experiment 2.

FIG. 4A is a graph illustrating the effect of dietary catalpicacid-supplementation on body weight in Experiment 2.

FIG. 4B is a graph illustrating the effect of dietary catalpicacid-supplementation on feed intake in Experiment 2.

FIG. 5 is a graph illustrating the effect of catalpicacid-supplementation on abdominal white adipose tissue weight on day 28of Experiment 2.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless otherwise stated, the following definitions are used throughoutthe present application:

Analysis of Variance (ANOVA): Arithmetic process for partitioning theoverall variation in data sets into specific components based on sourcesof variation. It has been used to determine whether numericaldifferences between treatment groups are statistically significant.

Adipogenesis: the process by which new adipocytes or fat storage cellsare generated.

Allele: one of a number of viable DNA codings of the same gene.

Conjugate triene: a polyunsaturated fatty acid containing three doublebonds separated by two single bonds.

Db/db mice: Term used to define a type of mouse which lacks both allelesof a long isoform of leptin receptor. This deficiency results in a highpredisposition to developing type 2 diabetes. Reference is made toExperiment 2 (infra.) for further discussions on Db/db mice.

Glycemia: concentration of glucose in blood.

Hyperglycemia: increased concentrations of glucose in blood beyond thenormal ranges.

Hyperinsulinemia: increased concentrations of insulin in blood beyondthe normal ranges.

Insulinemia: concentration of insulin in blood.

Insulin resistance: inability of tissues to respond to insulin and takeup glucose from the blood.

Substantially pure: having a purity of at least 90% by weight,preferably at least 95% by weight such as at least 98%, 99% or about100% by weight.

Type 2 diabetes or Non-insulin dependent Diabetes Mellitus: termreferring to a common type of diabetes caused by an unresponsiveness ofcells to the actions of insulin. If the cells do not respond to insulin,they are unable to take up glucose from blood, which results inglucotoxicity. In addition, the cells are deprived from the energyderived from glucose oxidation.

Catalpic Acid

The term as used herein refers to a conjugated linolenic acid isomercontaining trans-9, trans-11, cis-13 double bonds in the C₁₈ carbonchain, its non-toxic salts, active esters, active isomers, activemetabolites, structural lipids containing catalpic acid, and mixturesthereof. Catalpic acid is found in the seed oil of Catalpa ovata,Catalpa speciosa, Catalpa bungei and Catalpa bignininoides. Catalpicacid constitutes approximately 60% of the oil of the catalpa seed.Non-toxic salts include, for example, alkyl esters having from 1 to 6carbon atoms in the alkyl group, as well as mono-, di- andtri-glycerides, and mixtures thereof. Active isomers of catalpic acidinclude geometrical isomers and its non-toxic salts, e.g., sodium,potassium, calcium and magnesium salts, and its active esters, e.g.,alkyl esters having from 1 to 6 carbon atoms in the alkyl group, as wellas mono-, di- and tri-glycerides, and mixtures thereof.

Catalpic acid may be a substantially pure single chemical compound or amixture of one or more catalpic acid compounds as defined above. Forexample, the catalpic acid may be in the form of an extract obtainableor obtained from Catalpa seed oil, either directly or following one ormore steps of purification.

The catalpic acid used in the described methods may be in a free acidform or bound chemically through ester linkages. In its natural form,catalpic acid is heat stable. Catalpic acid may be used in its naturaloil state or in a dried and powdered form. Further, the free acid formof catalpic acid may be converted into a non-toxic salt, such as sodium,potassium or calcium salts, by reacting chemically equivalent amounts ofthe free acid form with an alkali hydroxide at a basic pH.

Administration

In the course of the method of the present invention, a therapeuticallyeffective amount of catalpic acid compound is administered to an animal,including mammals and humans, in many ways. While in the preferredembodiment, the catalpic acid compound is administered orally orparenterally, other forms of administration such as through medicalcompounds or aerosols are also contemplated.

For oral administration, the effective amount of catalpic acid may beadministered in, for example, a solid, semi-solid, liquid or gas state.Specific examples include tablet, capsule, powder, granule, solution,suspension, syrup, and elixir agents. However, the catalpic acidcompound is not limited to these forms.

To formulate the catalpic acid of the present invention into tablets,capsules, powders, granules, solutions or suspensions, the catalpic acidcompound is preferably mixed with a binder, a disintegrating agentand/or a lubricant. If necessary, the resultant composition may be mixedwith a diluent, a buffer, an infiltrating agent, a preservative and/or aflavor, using known methods. Examples of the binder include crystallinecellulose, cellulose derivatives, cornstarch, and gelatin. Examples ofthe disintegrating agent include cornstarch, potato starch, and sodiumcarboxymethylcellulose. Examples of the lubricant include talc andmagnesium stearate. Further, additives, which have been conventionallyused, such as lactose and mannitol, may also be used.

For parenteral administration, the catalpic acid compound of the presentinvention may be administered rectally or by injection. For rectaladministration, a suppository may be used. The suppository may beprepared by mixing the catalpic acid of the present invention with apharmaceutically suitable excipient that melts at body temperature butremains solid at room temperature. Examples include but are not limitedto cacao butter, carbon wax or polyethylene glycol. The resultingcomposition may be molded into any desired form using methods known tothe field.

For administration by injection, the catalpic acid compound of thepresent invention may be injected hypodermically, intracutaneously,intravenously or intramuscularly. Medicinal drugs for such injection maybe prepared by dissolving, suspending or emulsifying the catalpic acidof the invention into an aqueous or non-aqueous solvent such asvegetable oil, glyceride of synthetic resin acid, ester of higher fattyacid, or propylene glycol by a known method. If desired, additives suchas a solubilizing agent, an osmoregulating agent, an emulsifier, astabilizer, or a preservative, which has been conventionally used mayalso be added.

For formulating the catalpic acid of the present invention intosuspensions, syrups or elixirs, a pharmaceutically suitable solvent maybe used.

The catalpic acid compound of the present invention may also be usedtogether with an additional compound having other pharmaceuticallysuitable activity to prepare a medicinal drug.

The catalpic acid of the present invention may also be administered inthe form of an aerosol or inhalant prepared by charging the catalpicacid in the form of a liquid or fine powder, together with a gaseous orliquid spraying agent and, if necessary, a known auxiliary agent such asan inflating agent, into a non-pressurized container such as an aerosolcontainer or a nebulizer. A pressurized gas of, for example,dichlorofluoromethane, propane or nitrogen may be used as the sprayingagent.

Catalpic acid may be administered to an animal, including mammals andhumans, in need thereof as a pharmaceutical or veterinary composition,such as tablets, capsules, solutions or emulsions. In a preferredembodiment of the invention, the free acid form of catalpic acid isadministered. However, administration of other forms of catalpic acid,including but not limited to esters thereof, pharmaceutically-suitablesalts thereof, metabolites thereof, and combinations thereof, in asingle dose or a multiple dose, are also contemplated by the presentinvention.

Catalpic acid may also be administered to an animal in need thereof as anutritional additive, either as a food or nutraceutical supplement.

The catalpic acid is preferably used and/or administered in the form ofa composition. Suitable compositions are, preferably, a pharmaceuticalcomposition, a foodstuff or a food supplement. These compositionsprovide a convenient form in which to deliver the catalpic acid.Compositions of the invention may comprise an antioxidant in an amounteffective to increase the stability of the catalpic acid with respect tooxidation.

The amount of catalpic acid that is administered in the method of theinvention or that is for administration in the use of the invention ispreferably from about 0.001 g to about 20 g (more preferably 0.1 g to 10g, such as 0.5 g to 5 g) of catalpic acid or derivative thereof per day.Suitable compositions can be formulated accordingly.

A preferred composition according to the invention is a foodstuff. Foodproducts (which term includes animal feed) preferably contain a fatphase, wherein the fat phase contains catalpic acid. The foodstuffs areoptionally used as a blend with a complementary fat. For example, theblend may comprise 0.3-95 wt %, preferably 2-80 wt %, most preferably5-40 wt % of catalpic acid and 99.7-5 wt %, preferably 98-20 wt %, mostpreferably 95-60 wt % of a complementary fat, for example selected from:cocoa butter, cocoa butter equivalents, palm oil or fractions thereof,palm kernel oil or fractions thereof, interesterified mixtures of saidfats or fractions thereof, or liquid oils, selected from: sunflower oil,high oleic sunflower oil, soybean oil, rapeseed oil, cottonseed oil,fish oil, safflower oil, high oleic safflower oil, maize oil andMCT-oils. Examples of suitable foodstuffs include those selected fromthe group consisting of margarines, fat continuous or water continuousor bicontinuous spreads, fat reduced spreads, confectionery productssuch as chocolate or chocolate coatings or chocolate fillings or bakeryfillings, ice creams, ice cream coatings, ice cream inclusions,dressings, mayonnaises, cheeses, cream alternatives, dry soups, drinks,cereal bars, sauces, snack bars, dairy products, clinical nutritionproducts and infant formulations.

Other examples of compositions are pharmaceutical compositions, such asin the form of tablets, pills, capsules, caplets, multi-particulatesincluding: granules, beads, pellets and micro-encapsulated particles;powders, elixirs, syrups, suspensions and solutions. Pharmaceuticalcompositions will comprise a pharmaceutically acceptable diluent orcarrier. Pharmaceutical compositions are preferably adapted foradministration parenterally, e.g., orally. Orally administrablecompositions may be in solid or liquid form and may take the form oftablets, powders, suspensions and syrups. Optionally, the compositionscomprise one or more flavoring and/or coloring agents.

Pharmaceutically acceptable carriers suitable for use in suchcompositions are well known in the art of pharmacy. The compositions ofthe invention may contain 0.1-99% by weight of catalpic acid. Thecompositions of the invention are generally prepared in unit dosageform. Preferably the unit dosage of catalpic acid is from 1 mg to 1000mg (more preferably from 100 mg to 750 mg). The excipients used in thepreparation of these compositions are the excipients known in the art.

Further examples of product forms for the composition are foodsupplements, such as in the form of a soft gel or a hard capsulecomprising an encapsulating material selected from the group consistingof gelatin, starch, modified starch, starch derivatives such as glucose,sucrose, lactose and fructose. The encapsulating material may optionallycontain cross-linking or polymerizing agents, stabilizers, antioxidants,light absorbing agents for protecting light-sensitive fills,preservatives and the like. Preferably, the unit dosage of catalpic acidin the food supplements is from 1 mg to 1000 mg (more preferably from100 mg to 750 mg).

Dose

The method of the present invention administers a therapeuticallyeffective amount of catalpic acid compound to an animal in need thereof.The effective amount of catalpic acid depends on the form of catalpicacid compound administered, the duration of the administration, theroute of administration, e.g., oral or parenteral, the age of the animaland the condition of the animal.

For instance, an amount of catalpic acid effective to enhance treat andprevent type 2 diabetes and obesity in an animal ranges from 10-10,000mg/kg/day. A preferred effective amount of catalpic acid is 100 to 5,000mg/kg/day, with a more preferred dose being 10 to 100 mg/kg/day. Aneffective amount of approximately 35 to 40 mg/kg/day of catalpic acid isalso envisioned by the method of the present invention, with 38mg/kg/day the preferred dose. The upper limit of the effective amount tobe administered is not critical, as catalpic acid is relativelynon-toxic as long as the recipient's diet contains the necessaryessential fatty acids.

The effective amount of catalpic acid is most effective in treating andpreventing metabolic disorders such as type 2 diabetes and obesity of ananimal when administered to an animal for periods ranging from about 7to 100 days, with a preferred period of 15 to 50 days, and a mostpreferred period of 32 to 42 days.

When the effective amount of the catalpic acid compound of the presentinvention is administered in a nutritional, medical or veterinarycomposition, the preferred dose ranges from about 0.01 to 2.0% wt/wt tothe food or nutraceutical product.

Preparation of Catalpic Acid

Solvent extraction methods are recommended to obtain good oil yields.Before solvent extraction, seeds are steam-heated to reduce enzymatichydrolysis and improving processing. After heating, seeds of catalpabeans are finely ground and used in a solvent extraction. Regular liquidsolvents such as hexane require soaking the seeds multiple times for upto 12 to 20 hours with stirring, filtering, and solvent evaporation.Alternatively, catalpic acid-enriched catalpa oil may be generated byCO₂ super critical extraction in methods known to the art.

Because the oil is more susceptible to oxidative processes when releasedfrom the seed, extraction is preferably performed under a nitrogenblanket to prevent contact with the air. Additionally, the oil isnitrogen-purged and stored with one or various antioxidants in the darkat 4° C. or at −20° C. for longer-term storage. Oil from catalpa seedsis expressed during this process in an amount by weight of 10% of theweight of the seeds.

The following non-limiting examples illustrate the invention and do notlimit its scope in any way. In the examples and throughout thisspecification, all percentages, parts and ratios are by weight unlessindicated otherwise.

EXPERIMENTS Experiment 1

Experiment 1 was designed to determine the effect of catalpic acid onthe development of obesity and type 2 diabetes induced by high fatdiets. Specifically, we investigated whether catalpic acid was able tonormalize impaired glucose tolerance, prevent hyperglycemia andhyperinsulinemia and attenuate abdominal fat accumulation in mice fedhigh fat diets.

Methods

In Western countries the Metabolic Syndrome or Syndrome X (i.e.,diabetes, obesity, cardiovascular disease, hypertension andhyperlipidemia) is on a steady rise. The development of nutrition-basedtherapeutic or preventive interventions using orally active, naturalcompounds is not only timely but also urgently needed. A total offorty-seven C57BL6 mice were used in experiment 1. Twenty-five mice werefed a control diet and twenty-three mice were fed a diet supplementedwith catalpic acid (0.6 g catalpic acid/100 g food). For the first 32days of the experiment all diets contained 7% fat, 0.02 totalcholesterol, and they obtained 14.5% of calories from fat by replacingcatalpic acid with linoleic acid (wt/wt basis) in the control diet. SeeTable 1.

TABLE 1 Composition of the Regular Diets¹. Ingredient Control DietCatalpic Diet Casein 200 200 L-Cystine 3 3 Corn Starch 397.486 397.486Maltodextrin 132 132 Sucrose 100 100 Cellulose 50 50 Mineral Mix(AIN-93)² 35 35 Vitamin Mix (AIN-93)³ 10 10 Choline Bitartrate 2.5 2.5tert-butylhydroquinone⁴ 0.014 0.014 Soybean oil 60 60 Linoleic acid 10 —Catalpa oil — 10 ¹Provides approximately 7% fat and 0.02 totalcholesterol and it obtains 14.5% of calories from fat. ²Supplied per kgof vitamin mix: 3 g nicotinic acid, 1.6 g calcium pantotenate, 0.7 gpyridoxine HCl, 0.6 g Thiamin HCl, 0.6 g riboflavin, 0.2 g folic acid,0.02 g D-biotin, 2.5 g vitamin B₁₂ (0.1% in mannitol), 15 g DL-alphatocopheryl acetate (500 IU/g), 0.8 g vitamin A palmitate (500,000 IU/g),0.2 gvitamin D₃ (cholecalciferol, 500,000 IU/g), 0.075 g vitamin K(phylloquinone), and 974.705 g sucrose. ³Supplied per kg of mineral mix:357 g calcium carbonate, 196 g potassium phosphate monobasic, 70.78 gpotassium citrate, 74 g sodium chloride, 46.6 g potassium sulfate, 24.3g magnesium oxide, 6.06 g ferric citrate, 1.65 g zinccarbonate, 0.63 gmanganous carbonate, 0.31 g cupric carbonate, 0.01 g potassium iodate,0.01025 g sodium selenate, 0.00795 g ammonium paramolybdate, 1.45 gsodium meta-silicate, 0.275 g chromium potassium sulfate, 0.0174 glithium chloride, 0.0815 g boric acid, 0.0635 g sodiumfluoride, 0.0318 gnickel carbonate, hydroxide, tetrahydrate, 0.0066 g ammonium vanadate,and 220.716 g sucrose. ⁴Antioxidant.

These diets are defined as regular diets and were formulated to beisocaloric between treatment groups. On day 32 of the experiment, twentymice within each group were fed high fat diets containing 19.6% fat,0.2% total cholesterol that obtained 40.1% of calories from fat byreplacing catalpic acid with lard (wt/wt basis) in the control high fatdiet. See Table 2.

TABLE 2 Composition of the High Fat Diets¹. Ingredient Control DietCatalpic Diet Casein 232 232 L-Cystine 3.0 3.0 DL-Methionine 3.5 3.5Corn Starch 137 137 Maltodextrin 150 150 Sucrose 162.595 162.595Cellulose 50 50 Cholesterol 1.9 1.9 Mineral Mix (AIN-93)² 40.60 40.60Calcium phosphate dibasic 4.64 4.64 Vitamin Mix (AIN-93)³ 16.24 16.24Choline Bitartrate 5 5 tert-butylhydroquinone⁴ 0.02 0.02 Vitamin K,phylloquinone 0.005 0.005 Soybean oil 30 30 Lard 163.5 153.5 Catalpa oil— 10 ¹Provides approximately 19.6% fat and 0.2% total cholesterol and itobtains 40.1% of kilocalories (Kcal) from fat (4.4 Kcal/g). Kcal densityis approximately 16% higher than typical AIN-93G-based diets. ²Suppliedper kg of vitamin mix: 3 g nicotinic acid, 1.6 g calcium pantotenate,0.7 g pyridoxine HCl, 0.6 g Thiamin HCl, 0.6 g riboflavin, 0.2 g folicacid, 0.02 g D-biotin, 2.5 g vitamin B₁₂ (0.1% in mannitol), 15 gDL-alpha tocopheryl acetate (500 IU/g), 0.8 g vitamin A palmitate(500,000 IU/g), 0.2 gvitamin D₃ (cholecalciferol, 500,000 IU/g), 0.075 gvitamin K (phylloquinone), and 974.705 g sucrose. ³Supplied per kg ofmineral mix: 357 g calcium carbonate, 196 g potassium phosphatemonobasic, 70.78 g potassium citrate, 74 g sodium chloride, 46.6 gpotassium sulfate, 24.3 g magnesium oxide, 6.06 g ferric citrate, 1.65 gzinc carbonate, 0.63 g manganous carbonate, 0.31 g cupric carbonate,0.01 gpotassium iodate, 0.01025 g sodium selenate, 0.00795 g ammoniumparamolybdate, 1.45 g sodium meta-silicate, 0.275 g chromium potassiumsulfate, 0.0174 g lithium chloride, 0.0815 g boric acid, 0.0635 g sodiumfluoride, 0.0318 g nickel carbonate, hydroxide, tetrahydrate, 0.0066 gammonium vanadate, and 220.716 g sucrose. ⁴Antioxidant.

The high fat diets were also formulated to be isocaloric betweentreatment groups. The remaining mice within each group (n=5) were fedthe regular diets. On day 78 of the experiment, mice were killed, bloodwas collected and immediately analyzed for fasting glucoseconcentrations by using the Accu-Check Instant Plus System (RocheDiagnostics Corporation, Indianapolis, Ind.) or stored for subsequentanalysis of insulin concentrations in plasma. Abdominal white adiposetissue and interscapular brown adipose tissue were collected, weighedand stored at −80° C. for RNA analyses. Liver, lungs, kidneys, pancreasand heart were examined for macroscopic abnormalities (gross lesions),fixed in phosphate-buffered formalin (10%) and processed forhistological evaluation. All specimens were generally labeled with thefollowing information: 1) mouse number; 2) date collected; 3) experimentnumber; 4) type of solvent; and 5) tissue type.

Glucose Tolerance Tests

A glucose tolerance test was conducted on day 78 of the experiment.Animals were fasted overnight (14 hours). Mice were injectedintraperitoneally with D-glucose (1 g/kg body weight) and blood sampleswere collected via the tail vein prior to the injection (time 0) and at15, 30 and 60 minutes following the injection.

Determination of Serum Insulin Concentrations.

Serum insulin concentrations were determined by using commerciallyavailable enzyme-linked immunosorbent assay kits (Linco Research, St.Charles, Mo.).

Statistics

Data were analyzed by ANOVA. The ANOVA was performed by using thegeneral linear model procedure of SAS (SAS Institute Inc., Cary, N.C.)as previously described (Bassaganya-Riera et al. 2004). Differences withprobability value (P<0.05) were considered significant.

Results

FIG. 1A illustrates the effect of catalpic acid on blood glucoseconcentrations during a glucose tolerance test in mice fed regular dietsfrom Experiment 1. Mice were fed either a control diet (filled squares)or a diet supplemented with catalpic acid (1 g/10 g; open rubies). FIG.1B illustrates the effect of catalpic acid on blood glucoseconcentrations during a glucose tolerance test in mice fed high fatdiets from Experiment 1. Mice were fed either a high fat control diet(filled squares) or a high fat diet supplemented with catalpic acid (1g/100 g; open rubies). Statistically significant differences (P<0.05)between treatments attributed to the main effects of the diet (*) areindicated.

Excessive abdominal fat accumulation and insulin resistance are keycharacteristics that typify the Metabolic Syndrome. The glucosetolerance tests are standard methods for evaluating glucose homeostasisin vivo. By using the glucose tolerance test, we discovered that glucosetolerance was not different between the two groups fed regular diets,which did not develop a diabetic phenotype (FIG. 1A). However, theability of mice fed a control high fat diet to normalize impairedglucose tolerance was significantly impaired when compared to those fedthe high fat diets supplemented with catalpic acid (FIG. 1B).

Furthermore, mice fed the control high fat diet were markedly morehyperglycemic and hyperinsulinemic than mice fed high fat dietssupplemented with catalpic acid or mice fed regular diets. Reference ismade to Table 3.

TABLE 3 Weight of abdominal white adipose tissue and interscapular brownadipose tissue and plasma fasting glucose and insulin concentrations inmice fed control or catalpic acid (CAT)-supplemented regular and highfat diets¹. Regular Diets High Fat Diets ANOVA Item Control CAT ControlCAT SEM² P value White 0.865^(b) 0.751^(b) 1.478^(a) 0.607^(b) 0.060.0001 adipose tissue, g Brown 0.130 0.169 0.129 0.105 0.008 0.08adipose tissue, g Glucose 153.8^(b) 146.20^(b) 301.30^(a) 205.43^(b)11.3 0.0001 (mg/dL) Insulin 0.932^(b) 0.960^(b) 2.254^(a) 0.730^(b) 0.230.01 (ng/mL) ¹Least squares means values in a row for a particulartissue with different superscripts are significantly different (P <0.05). ²Pooled standard error of the least square means.

Thus, catalpic acid-supplementation prevents or ameliorates thedevelopment of hyperglycemia, attenuates the hyperinsulinemia andnormalizes impaired glucose tolerance in mice fed high fat diets (Table3). These findings are clinically significant in the prevention andtreatment of type 2 diabetes, the Metabolic Syndrome and theircomplications (e.g., cardiovascular disease, stroke, retinopathy,nephropathy, and amputations).

The hyperglycemia and hyperinsulinemia observed in mice fed control highfat diets correlated with increased abdominal white adipose tissuedeposition (Table 3). However, no differences in brown adipose tissueweights were observed between groups. The decreased abdominal adiposityobserved in mice fed high fat diets supplemented with catalpic acid whencompared with mice fed the control diet could be caused by eithersuppressed adipogenesis or increased fatty acid consumption. Becausefatty liver or enlarged viscera were not observed in mice fed catalpicacid-supplemented diets, the decreased abdominal obesity is unlikely tobe caused by decreased adipogenesis and it may be due to increased fattyacid consumption. All of which, suggests that catalpic acid could beutilized in the treatment and prevention of insulin resistance,abdominal obesity and the Metabolic Syndrome.

Experiment 2

Objective

The data in Experiment 1 demonstrated that dietary catalpicacid-supplementation for 78 days had a positive effect on glucosetolerance, insulin resistance and abdominal adiposity. The goal of thisstudy was to investigate whether dietary catalpic acid-supplementationfor 30 days ameliorated or prevented impaired glucose tolerance, insulinresistance and abdominal adiposity in type 2 diabetic mice.

Methods

Mice were purchased from Harlan (Indianapolis, Ind.). Following anacclimation period of 5 days on regular rodent chow they were fedpurified, high fat diets that represented a modification of the AIN-93Grodent diet in which the nutritional requirements, including those forpolyunsaturated fatty acids (PUFA), were met or exceeded. See Table 4.

TABLE 4 Composition of the High Fat Diets in Experiment 2¹. Ingredient(g/kg) Control Diet CAT Diet Casein 232 232 L-Cystine 3.0 3.0DL-Methionine 3.5 3.5 Corn Starch 137 137 Maltodextrin 150 150 Sucrose162.58 162.58 Cellulose 50 50 Cholesterol 1.9 1.9 Mineral Mix (AIN-93)²40.60 40.60 Calcium phosphate dibasic 4.64 4.64 Vitamin Mix (AIN-93)³16.24 16.24 Choline Bitartrate⁴ 5 5 tert-butylhydroquinone⁵ 0.04 0.04Soybean oil 30 30 Lard 153.5 153.5 Soybean oil 10 — Catalpic oil — 10¹Provides approximately 19.6% fat, 0.2% total cholesterol and 4.4kilocalories/g (kcal/g) it obtains 40% kcal from fat. Kcal density isapproximately 16% higher than typical AIN-93G-based diets. ²Supplied perkg of vitamin mix: 3 g nicotinic acid, 1.6 g calcium pantotenate, 0.7 gpyridoxine HCl, 0.6 g Thiamin HCl, 0.6 g riboflavin, 0.2 g folic acid,0.02 g D-biotin, 2.5 g vitamin B₁₂ (0.1% in mannitol), 15 g DL-alphatocopheryl acetate (500 IU/g), 0.8 g vitamin A palmitate (500,000 IU/g),0.2 gvitamin D₃ (cholecalciferol, 500,000 IU/g), 0.075 g vitamin K(phylloquinone), and 974.705 g sucrose. ³Supplied per kg of mineral mix:357 g calcium carbonate, 196 g potassium phosphate monobasic, 70.78 gpotassium citrate, 74 g sodium chloride, 46.6 g potassium sulfate, 24.3g magnesium oxide, 6.06 g ferric citrate, 1.65 g zinc carbonate, 0.63 gmanganous carbonate, 0.31 g cupric carbonate, 0.01 gpotassium iodate,0.01025 g sodium selenate, 0.00795 g ammonium paramolybdate, 1.45 gsodium meta-silicate, 0.275 g chromium potassium sulfate, 0.0174 glithium chloride, 0.0815 g boric acid, 0.0635 g sodium fluoride, 0.0318g nickel carbonate, hydroxide, tetrahydrate, 0.0066 g ammonium vanadate,and 220.716 g sucrose. ⁴The choline bitartrate concentrations have beenincreased from 2.5 g/kg in regular AIN-93G diets to 5 g/kg due toincreased kcal density of high fat diets. ⁵Antioxidant.

Stock fatty acid solutions, e.g., soybean oil or catalpic acid were keptat −20° C. and nitrogen-purged every time that the bottles were opened.All the experimental diets contained the same amount of energy(isocaloric) and protein (isonitrogenous). To maintain the dietsisocaloric, catalpic acid replaced soybean oil in the catalpic aciddiet. We utilized male, db/db [BKS.Cg−+Lepr^(db)/+Lepr^(db)/OlaHsd (Type2 Diabetic)] mice (n=20) weighing 19.86 g on day 1 of the study. Thedb/db mice lack the long isoform of leptin receptor and represent awell-established model of Type 2 Diabetes and are often utilized todetermine the pre-clinical efficacy of antidiabetic drugs. Half of themice were fed a control and the other half a catalpic acid-supplementeddiet (1 g catalpa oil/100 g diet).

Assessment of Type 2 Diabetes

All mice were determined normoglycemic (210 mg/dl glucose or lower) andof similar body weights prior to assignment to experimental treatments.Mice were weighed on a daily basis and examined for clinical signs ofdisease by blinded observers. Water and feed intake were assessed on adaily basis. Fasting (12 h) glucose and insulin concentrations weredetermined on days 0, 7, 15, 21 and 28 of the study. Briefly, blood wascollected via the caudal vein and placed onto capillary blood collectiontubes. Insulin concentrations were determined by using a commerciallyavailable insulin enzyme-linked immunosorbent assay (ELISA) (LincoResearch, St Charles, Mo.) per manufacturer's instructions. Mice wereadministered a glucose tolerance test and euthanized by CO₂ narcosis onday 28 of the study. For the glucose tolerance test, animals were fastedovernight (12 hours), injected intraperitoneally with D-glucose (2 g/kgbody weight) and blood samples were collected via the caudal vein priorto the injection (time 0) and at 15, 30, 60, 90, 120 and 180 minutesfollowing the injection to determine the effect of CAT on the kineticsof glucose normalization.

Statistics

Data were analyzed by ANOVA. The ANOVA was performed by using thegeneral linear model procedure of SAS (SAS Institute Inc., Cary, N.C.)as previously described (Bassaganya-Riera, Reynolds et al. 2004). Datawere analyzed as a completely randomized design. Differences withprobability value (P<0.05) were considered significant.

Results

Fasting Glucose and Insulin Concentrations

FIG. 2A illustrates the effect of dietary catalpic acid-supplementationon fasting plasma glucose concentrations on days 0, 7, 14, 21 and 28 ofExperiment 2. Db/db mice fed either a high fat control diet (filledsquares) or a high fat diet supplemented with catalpic acid (1 g/100 g;open rubies). Statistically significant differences (P<0.05) betweentreatments attributed to the main effects of the diet (*) are indicated.FIG. 2B illustrates the effect of dietary catalpic acid-supplementationon plasma glucose concentrations during a glucose tolerance test fromExperiment 2. Blood was collected at 0, 15, 30, 60, 90, 120 and 180minutes following the intraperitoneal glucose challenge (2 g/Kg bodyweight). Db/db mice fed either a high fat control diet (filled squares)or a high fat diet supplemented with catalpic acid (1 g/10 g; openrubies). Statistically significant differences (P<0.05) betweentreatments attributed to the main effects of the diet (*) are indicated.

To determine the effect of catalpic acid on insulin sensitivity andglucose homeostasis, we examined fasting plasma glucose and insulinconcentrations on days 0, 7, 14, 21 and 28 of the study. We found thatthe levels of plasma glucose were significantly (P<0.05) lower in micefed high fat, catalpic acid-supplemented diets than in those fed highfat control diets on days 14, 21 and 28 (FIG. 2A). On day 28 of theexperiment the glucose concentrations in db/db mice fed the control highfat diet corresponded to a diabetic phenotype (425 mg/dl) whereasdietary catalpic acid-supplementation maintained mice in a pre-diabeticstage (290 mg/dl) (FIG. 2B).

These differences in plasma glucose levels observed on day 28 of thestudy in db/db mice correlated with plasma insulin concentrations asshown in FIG. 3. FIG. 3 illustrates the effect of dietary catalpicacid-supplementation on fasting plasma insulin concentrations on days 0,7, 14 and 28 of Experiment 2. Db/db mice fed either a high fat controldiet (filled squares) or a high fat diet supplemented with catalpic acid(1 g/100 g; open rubies). Statistically significant differences (P<0.05)between treatments attributed to the main effects of the diet (*) areindicated. More specifically, plasma insulin concentrations in db/dbmice fed the catalpic acid-supplemented diets remained at basal levels(i.e., lower than 4 ng/ml). However, db/db mice fed the control dietoverproduced insulin on day 7 (6.54 ng/ml) and the insulin levels inmice fed the control diet remained higher throughout the study (FIG. 3).

Glucose Tolerance Test

To determine whether dietary catalpic acid would enhance the glucosenormalizing ability of mice fed high fat diets, we performed anintraperitoneal glucose challenge and evaluated the kinetics of plasmaglucose at 0, 15, 30, 60, 90, 120 and 180 minutes following the glucoseinjection. We found that the glucose normalizing ability of mice fedcatalpic acid-supplemented diets was greater than in mice fed controldiets in all time points examined as shown in FIG. 2B.

Body Weight, Feed Intake and White Adipose Tissue (WAT) Weight

FIG. 4A illustrates the effect of dietary catalpic acid-supplementationon body weight in Experiment 2. Mice were weighed on a daily basis.Db/db mice fed either a high fat control diet (filled squares) or a highfat diet supplemented with catalpic acid (1 g/100 g; open rubies).Statistically significant differences (P<0.05) between treatmentsattributed to the main effects of the diet (*) are indicated. FIG. 4Billustrates the effect of dietary catalpic acid-supplementation on feedintake in Experiment 2. Feeders and food were weighed on a daily basis.Db/db mice fed either a high fat control diet (filled squares) or a highfat diet supplemented with catalpic acid (1 g/100 g; open rubies).Statistically significant differences (P<0.05) between treatmentsattributed to the main effects of the diet (*) are indicated.

No differences were found in body weight (FIG. 4A) or feed intake (FIG.4B). Thus, catalpic acid did not elicit the side effects of currentantidiabetic medications. Even though there were no differences in bodyweight, the WAT weight of mice fed a catalpic acid-supplemented diet wassignificantly lower than that recovered from mice fed a control dietwhich is shown in FIG. 5. FIG. 5 illustrates the effect of catalpicacid-supplementation on abdominal white adipose tissue weight on day 28of Experiment 2. Db/db mice fed either a high fat control diet (filledbar) or a high fat diet supplemented with catalpic acid (1 g/100 g; openbar). Statistically significant differences (P<0.05) between treatmentsattributed to the main effects of the diet (*) are indicated. Thedifference in WAT weight between groups attributable to catalpic acidwas over 10%.

The glucose tolerance tests are standard methods for evaluating glucosehomeostasis in vivo. By using the glucose tolerance test, we previouslydiscovered that glucose tolerance was not different between mice fedregular diets, which did not develop a diabetic phenotype. However, theability of mice fed a control high fat diet to normalize plasma glucoseconcentrations was significantly impaired when compared to those fed thehigh fat diets supplemented with catalpic acid (Experiment 1). Herein,we have confirmed that dietary catalpic acid-supplementation amelioratesthe glucose intolerance induced by high fat, diabetogenic diets in mice.In addition, in our previous studies catalpic acid was fed for 78 days,whereas here we observed an effect of dietary catalpic acid after 7 to28 days of dietary supplementation.

Fasting glucose and insulin concentrations in plasma represent goodindicators of insulin sensitivity. Experiment 1 demonstrated that plasmaglucose and insulin concentrations were lower in mice fedcatalpic-supplemented, high fat diets than in control, high fat diets.Herein, we have confirmed our previous findings. In addition we havedemonstrated that catalpic had an immediate effect on insulinconcentrations (i.e. 7 days). More specifically, mice fed catalpicacid-supplemented diets were capable of better overcoming the initialmetabolic response to the high fat diet. In addition, this initialbeneficial effect of dietary catalpic acid-supplementation wasmaintained throughout the study.

All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to certain preferred embodiments thereof,and many details have been set forth for purposes of illustration, itwill be apparent to those skilled in the art that the invention issusceptible to additional embodiments and that certain of the detailsdescribed herein may be varied considerably without departing from thebasic principles of the invention.

BIBLIOGRAPHY

-   Bassaganya-Riera, J., K. Reynolds, et al. (2004). “Activation of    PPAR gamma and delta by conjugated linoleic acid mediates protection    from experimental inflammatory bowel disease.” Gastroenterology    127(3): 777-91.-   Center for Disease Control (2004). National diabetes fact sheet:    general information and national estimates on diabetes in the United    States 2003. Atlanta, U.S. Department of Health and Human Services,    Centers for Disease control and Prevention.-   Ginsberg, H. N. (2003). “Treatment for patients with the metabolic    syndrome.” Am J Cardiol 91(7A): 29E-39E.-   Wang, Y. X., C. H. Lee, et al. (2003).    “Peroxisome-proliferator-activated receptor delta activates fat    metabolism to prevent obesity.” Cell 113(2): 159-70.-   U.S. Pat. No. 6,451,439 to Okamoto-   U.S. Pat. No. 6,593,514 to Cahoon-   U.S. Patent Application 20030126640 to Cahoon-   U.S. Patent Application 20020045232 to Qiu

1. A method of treating type 2 diabetes and abdominal obesity signscomprising administering a composition, wherein the composition consistsessentially of an effective amount of a compound selected from the groupconsisting of catalpic acid in its free acid form, non-toxic saltsthereof, lipids containing catalpic acid, and mixtures thereof, incombination with a pharmaceutically-acceptable carrier, wherein theeffective amount of the compound is between about 0.001 g to about 20 gper day and the pharmaceutically acceptable carrier is in the form ofcapsules, cachets, tablets, boluses or lozenges.
 2. The method of claim1, comprising administering a free form of catalpic acid.
 3. The methodof claim 1, comprising administering the compound orally in combinationwith a pharmaceutically suitable oral carrier.
 4. The method of claim 3wherein the compound is administered orally in a solid, semi-solid,liquid or gas state.
 5. The method of claim 1, comprising administeringthe compound parenterally.
 6. The method of claim 1, wherein thecompound is administered in combination with one or more vitamins orfatty acids.
 7. The method of claim 1 for the treatment ofhyperinsulinemia.
 8. The method of claim 1 for the treatment ofhyperglycemia.
 9. The method of claim 1 for the treatment of abdominalfat accumulation.
 10. The method of claim 1 for the treatment ofimpaired glucose tolerance.
 11. The method of claim 1, wherein theeffective amount of the compound is between about 0.1 g to about 10 gper day.
 12. A composition for treating type 2 diabetes and abdominalobesity signs consisting essentially of an effective amount of acompound selected from the group consisting of catalpic acid in its freeacid form, non-toxic salts thereof, lipids containing catalpic acid, andmixtures thereof, in combination with a pharmaceutically-acceptablecarrier, wherein the effective amount of the compound is between about0.001 g to about 20 g per day and the pharmaceutically acceptablecarrier is in the form of capsules, cachets, tablets, boluses orlozenges.
 13. The composition of claim 12 wherein the pharmaceuticallyacceptable carrier is in a form suitable for parenteral administration.14. The composition of claim 12 wherein the pharmaceutically acceptablecarrier is in a form suitable for oral administration.
 15. Thecomposition of claim 12, wherein the effective amount of the compound isbetween about 0.1 g to about 10 g per day.